Detection of nucleic acids and production of type I interferons (IFNs) are principal elements of antiviral defense, but can cause autoimmunity if misregulated. In previous work, we discovered the interferon- stimulatory DNA (ISD) pathway, a cell-intrinsic antiviral response that is activated by detection of cytosolic DNA. We then identified 3' repair exonuclease 1 (Trex1) as a cytosolic, ISD-binding DNA exonuclease. Remarkably, loss of function mutations in the human Trex1 gene cause Aicardi-Goutieres Syndrome (AGS), a rare and severe disease that resembles congenitally acquired viral infection. Using Trex1-deficient mice as a model of AGS, we defined Trex1 as an essential negative regulator of the ISD pathway, thus revealing a novel, cell-intrinsic mechanism for initiation of autoimmunity. Specifically, accumulation of endogenous Trex1 DNA substrates within cells triggers type I IFN production through the ISD pathway, which then leads to the activation and recruitment of autoreactive lymphocytes that attack the initiating cells. Our findings raise fundamental new questions about the connections between the innate antiviral response and autoimmunity, with important implications for the treatment of AGS and related diseases. Very little is known about the cells that initiate disease, the source and nature of the DNA that accumulates within these initiating cells, or how detection of this accumulated DNA ultimately links to the autoreactive lymphocyte response. In this proposal, we will develop new tools and apply a combination of genetics, molecular biology, biochemistry, and cellular immunology to address these important questions. In Aim 1, we will apply new genetic tools to visualize the in vivo dynamics of the IFN-dependent autoimmune response with unprecedented resolution. In Aim 2, we will identify and characterize the Trex1 DNA substrates that cause disease. In Aim 3, we will define how cell- intrinsic nucleic acid detection is linked to the adaptive immune response. Our long term goals are twofold: we will rigorously define the basis of disease with the hope of developing new treatments and preventative measures to cure AGS and related diseases, and we will use this model system to probe more general questions about interconnections between innate immune detection of nucleic acids and lymphocyte-mediated antiviral responses.